Magnet-Guided Temozolomide and Ferucarbotran Loaded Nanoparticles to Enhance Therapeutic Efficacy in Glioma Model.

Background. The aim of the study was to synthesize liposomal nanoparticles loaded with temozolomide and ferucarbotran (LTF) and to evaluate the theranostic effect of LTF in the glioma model. Methods. We synthesized an LTF that could pass through the Blood Brain Barrier (BBB) and localize in brain tumor tissue with the help of magnet guidance. We examined the chemical characteristics. Cellular uptake and cytotoxicity studies were conducted in vitro. A biodistribution and tumor inhibition study was conduted using an in vivo glioma model. Results. The particle size and surface charge of LTF show 108 nm and -38 mV, respectively. Additionally, the presence of ferucarbotran significantly increased the contrast agent effect of glioma compared to the control group in MR imaging. Magnet-guided LTF significantly reduced the tumor size compared to control and other groups. Furthermore, compared to the control group, our results demonstrate a significant inhibition in brain tumor size and an increase in lifespan. Conclusions. These findings suggest that the LTF with magnetic guidance represents a novel approach to address current obstacles, such as BBB penetration of nanoparticles and drug resistance. Magnet-guided LTF is able to enhance therapeutic efficacy in mouse brain glioma.

The long multi-epitope peptide vaccine combined with adjuvants improved the therapeutic effects in a glioblastoma mouse model.

Emerging data have suggested that single short peptides have limited success as a cancer vaccine; however, extending the short peptides into longer multi-epitope peptides overcame the immune tolerance and induced an immune response. Moreover, the combination of adjuvants such as lenalidomide and anti-programmed cell death protein 1 (PD1) with a peptide vaccine showed potential vaccine effects in previous studies. Therefore, the effects of a long multi-epitope peptide vaccine in combination with lenalidomide and anti-PD1 were analyzed in this study. Long multi-epitope peptides from two MHCI peptides (BIRC597-104 and EphA2682-689) and the pan-human leukocyte antigen-DR isotype (HLA-DR) binding epitope (PADRE) were synthesized. The therapeutic effects of long multi-epitope peptides in combination with lenalidomide and anti-PD1 were confirmed in the murine GL261 intracranial glioma model. Immune cells' distribution and responses to the long multi-epitope peptides in combination with these adjuvants were also estimated in the spleens, lymph nodes, and tumor tissues. The difference between long multi-epitope peptides and a cocktail of multi-epitope peptides combined with lenalidomide and anti-PD1 was also clarified. As a result, long multi-epitope peptides combined with lenalidomide and anti-PD1 prolonged the survival of mice according to the suppression of tumor growth in an intracranial mouse model. While long multi-epitope peptides combined with these adjuvants enhanced the percentages of activated and memory effector CD8+ T cells, the increase in percentages of regulatory T cells (Tregs) was observed in a cocktail of multi-epitope peptides combined with lenalidomide and anti-PD1 group in the tumors. Long multi-epitope peptides combined with these adjuvants also enhanced the function of immune cells according to the enhanced pro-inflammatory cytokines and cytotoxicity against GL261 cells in ex vivo. In conclusion, long multi-epitope peptides composed of MHCI peptides, BIRC5 and EphA2, and the MHCII peptide, PADRE, in combination with lenalidomide and anti-PD1 has the potential to improve the therapeutic effects of a vaccine against GBM.

Natural killer cell therapy potentially enhances the antitumor effects of bevacizumab plus irinotecan in a glioblastoma mouse model.

Various combination treatments have been considered to attain the effective therapy threshold by combining independent antitumor mechanisms against the heterogeneous characteristics of tumor cells in malignant brain tumors. In this study, the natural killer (NK) cells associated with bevacizumab (Bev) plus irinotecan (Iri) against glioblastoma multiforme (GBM) were investigated. For the experimental design, NK cells were expanded and activated by K562 cells expressing the OX40 ligand and membrane-bound IL-18 and IL-21. The effects of Bev and Iri on the proliferation and NK ligand expression of GBM cells were evaluated through MTT assay and flow cytometry. The cytotoxic effects of NK cells against Bev plus Iri-treated GBM cells were also predicted via the LDH assay in vitro. The therapeutic effect of different injected NK cell routes and numbers combined with the different doses of Bev and Iri was confirmed according to tumor size and survival in the subcutaneous (s.c) and intracranial (i.c) U87 xenograft NOD/SCID IL-12Rγnull mouse model. The presence of injected-NK cells in tumors was detected using flow cytometry and immunohistochemistry ex vivo. As a result, Iri was found to affect the proliferation and NK ligand expression of GBM cells, while Bev did not cause differences in these cellular processes. However, the administration of Bev modulated Iri efficacy in the i.c U87 mouse model. NK cells significantly enhanced the cytotoxic effects against Bev plus Iri-treated GBM cells in vitro. Although the intravenous (IV) injection of NK cells in combination with Bev plus Iri significantly reduced the tumor volume in the s.c U87 mouse model, only the direct intratumorally (IT) injection of NK cells in combination with Bev plus Iri elicited delayed tumor growth in the i.c U87 mouse model. Tumor-infiltrating NK cells were detected after IV injection of NK cells in both s.c and i.c U87 mouse models. In conclusion, the potential therapeutic effect of NK cells combined with Bev plus Iri against GBM cells was limited in this study. Accordingly, further research is required to improve the accessibility and strength of NK cell function in this combination treatment.

Branched Multipeptide-combined Adjuvants Potentially Improve the Antitumor Effects on Glioblastoma.

The promising immunotherapy effects of a multiple antigenic peptide on glioblastoma (GBM) in a previous study encourage the use of adjuvants to enhance its therapeutic efficacy. Among adjuvants, pan HLA-DR-binding epitope (PADRE) and anti-programmed cell death protein 1 (anti-PD1) have potentially been tested for cancer immunotherapy. Therefore, here we evaluated the ability of PADRE and anti-PD1 to enhance the function of the branched multipeptide against GBM. The potential utility of tumor-associated antigens (ErbB-2 and WT-1) targeting GBM with HLA-A24 was confirmed and a branched multipeptide was constructed from these antigens. The effects of the branched multipeptide and PADRE on immunophenotyping and polarized Th cytokine production in dendritic cells were clarified. The expression of PD1 on T cells and PDL1 on GBM cells was also investigated. The interferon-γ enzyme-linked immunospot and lactate dehydrogenase release assays were performed to determine the function of GBM peptide antigen-specific cytotoxic T cells against GBM cells. Overall, this study showed that both ErbB-2 and WT-1 are potential candidates for branched multipeptide construction. The branched multipeptide and PADRE enhanced the expression of major histocompatibility complex and co-stimulatory molecules and the production of polarized Th1 cytokines in dendritic cells. The increase in the number of interferon-γ+ effector T cells was consistent with the increase in the percentage specific lysis of GBM target cells by GBM peptide antigen-specific cytotoxic T cells in the presence of the branched multipeptide, PADRE, and anti-PD1. Our study suggests the combination of branched multipeptide and adjuvants such as PADRE and anti-PD1 can potentially enhance the effects of immunotherapy for GBM treatment.

Peptide Vaccine Combined Adjuvants Modulate Anti-tumor Effects of Radiation in Glioblastoma Mouse Model.

Glioblastoma, the most common aggressive cancer, has a poor prognosis. Among the current standard treatment strategies, radiation therapy is the most commonly recommended. However, it is often unsuccessful at completely eliminating the cancer from the brain. A combination of radiation with other treatment methods should therefore be considered. It has been reported that radiotherapy in combination with immunotherapy might show a synergistic effect; however, this still needs to be investigated. In the current study, a "branched multipeptide and peptide adjuvants [such as pan DR epitope (PADRE) and polyinosinic-polycytidylic acid-stabilized with polylysine and carboxymethylcellulose-(poly-ICLC)]," namely vaccine and anti-PD1, were used as components of immunotherapy to assist in the anti-tumor effects of radiotherapy against glioblastomas. With regard to experimental design, immunological characterization of GL261 cells was performed and the effects of radiation on this cell line were also evaluated. An intracranial GL261 mouse glioma model was established, and therapeutic effects were observed based on tumor size and survival time. The distribution of effector immune cells in the spleen, based on cytotoxic T lymphocyte (CTL) and natural killer (NK) cell function, was determined. The pro-inflammatory and anti-inflammatory cytokine production from re-stimulated splenocytes and single tumor cells were also evaluated. As GL261 cells demonstrated both immunological characteristics and radiation sensitivity, they were found to be promising candidates for testing this combination treatment. Combinatorial treatment with radiation, vaccine, and anti-PD1 prolonged mouse survival by delaying tumor growth. Although this combination treatment led to an increase in the functional activity of both CTLs and NK cells, as evidenced by the increased percentage of these cells in the spleen, there was a greater shift toward CTL rather than NK cell activity. Moreover, the released cytokines from re-stimulated splenocytes and single tumor cells also showed a shift toward the pro-inflammatory response. This study suggests that immunotherapy comprising a branched multipeptide plus PADRE, poly-ICLC, and anti-PD1 could potentially enhance the anti-tumor effects of radiotherapy in a glioblastoma mouse model.

Feasibility of dendritic cell-based vaccine against glioblastoma by using cytoplasmic transduction peptide (CTP)-fused protein antigens combined with anti-PD1.

Recent clinical trials utilizing antigen-pulsed dendritic cells (DCs) have demonstrated increased survival of vaccinated cancer patients. Besides, the cytoplasmic transduction peptide (CTP) not only has an excellent transcellular efficiency but also shows a strong tendency to remain in the cytoplasm after transduction, without migrating into the nucleus. In this study, we investigated the effectiveness of immunotherapy against malignant gliomas using DCs pulsed with CTP-fused protein antigens combined with programmed cell death protein 1 blockade (anti-PD1). The expression of tumor associated antigen (WT1 and BIRC5) and PDL1 on glioblastoma (GBM) target cells was confirmed by western blot. The effect of CTP-fused protein antigens on mature DCs (VaxDCs) was determined. The immunophenotypes of VaxDCs pulsed with CTP-fused protein antigens was confirmed by flow cytometry and the cytokine production levels of T helper polarization were measured by enzyme-linked immunosorbent (ELISA) assay. The IFN-γ-enzyme linked immunospot and lactate dehydrogenase release assays were performed to estimate the cytotoxic activity of antigen-specific cytotoxic T lymphocytes (CTLs), stimulated by VaxDCs pulsed with CTP-fused protein antigens and anti-PD1, against malignant glioma cells expressing target antigens. VaxDCs pulsed with CTP-fused protein antigens showed enhanced expression of major histocompatibility complex (MHC) and co-stimulatory markers of DCs and resulted in Th1 cytokine polarization. The increase in the number of IFN-γ+ effector T cells paralleled with the enhanced percent specific lysis of GBM targets cells by antigen-specific CTLs. Our study suggested that using CTP-fused protein antigens for DC vaccine preparation along with PD1 blockade could be an effective immunotherapy strategy for GBM.

Branched multipeptide immunotherapy for glioblastoma using human leukocyte antigen-A*0201-restricted cytotoxic T-lymphocyte epitopes from ERBB2, BIRC5 and CD99.

We investigated the use of cytotoxic T-lymphocyte (CTL) epitopes in peptide immunotherapy for glioblastoma. Three peptides (ERBB2, BIRC5 and CD99) were selected based on their peptide-T2 cell binding affinities and combined in a multipeptide cocktail or a branched multipeptide synthesized with mini-polyethylene glycol spacers. Dendritic cells (DCs) pulsed with the multipeptide cocktail or branched multipeptide were compared based on their immunophenotype and cytokine secretion. FACS analysis of alpha-type 1 polarized dendritic cells (αDC1s) revealed that both groups highly expressed CD80, CD83 and CD86, indicating that both treatments efficiently generated mature αDC1s with the expected phenotype. Production of IL-12p70, IL-12p40 and IL-10 also increased upon αDC1 maturation in both groups. CTLs stimulated by either αDC1 group ("DC-CTLs") included numerous IFN-γ-secreting cells against T2 cells loaded with the corresponding multipeptides. Large numbers of IFN-γ-secreting cells were observed when human glioblastoma cell lines and primary cells were treated with multipeptide-pulsed DC-CTLs. Both multipeptide-pulsed DC-CTL groups exhibited cytotoxic activity of 40-60% against the U251 cell line and 60-80% against primary cells. Branched multipeptide from ERBB2, BIRC5 and CD99 stably bound with T2 cells, and its cytotoxicity toward target cells was similar to that of the multipeptide cocktail. Thus, branched multipeptides could be promising candidates for immunotherapeutic glioblastoma treatment.